A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Abstract

Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it. Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it. Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1Hubner C.A. Jentsch T.J. Hum. Mol. Genet. 2002; 11: 2435-2445Crossref PubMed Scopus (182) Google Scholar). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na+ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2George Jr., A.L. Epilepsy Curr. 2004; 4: 65-70Crossref PubMed Scopus (44) Google Scholar, 3Jurkat-Rott K. Lerche H. Lehmann-Horn F. J. Neurol. 2002; 249: 1493-1502Crossref PubMed Scopus (108) Google Scholar, 4Kass R.S. Moss A.J. J. Clin. Invest. 2003; 112: 810-815Crossref PubMed Scopus (121) Google Scholar). Clinical consequences of inherited mutations that disrupt Na+ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins. In the heart, Na+ channels (NaV1.5) 4The abbreviations used are: NaV1.5, voltage-gated sodium channel isoform 1.5; GST, glutathione S-transferase; ISUS, sustained current; WT, wild type; TTX, tetrodotoxin; HEK, human embryonic kidney. primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for NaV1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5Clancy C.E. Kass R.S. Physiol. Rev. 2005; 85: 33-47Crossref PubMed Scopus (81) Google Scholar), and in most cases, these inherited mutations disrupt channel inactivation. Fast inactivation of Na+ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization (6Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar). Inherited mutations of the III/IV linker in the cardiac Na+ channel can disrupt fast inactivation, resulting in sustained current (ISUS), which can cause long QT syndrome variant 3 (5Clancy C.E. Kass R.S. Physiol. Rev. 2005; 85: 33-47Crossref PubMed Scopus (81) Google Scholar). However, the NaV1.5 COOH terminus also has been shown to play a role in inactivation both through chimeric studies (7Mantegazza M. Yu F.H. Catterall W.A. Scheuer T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15348-15353Crossref PubMed Scopus (108) Google Scholar), through the characterization of several disease-linked mutations found in the C terminus (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 11An R.H. Wang X.L. Kerem B. Benhorin J. Medina A. Goldmit M. Kass R.S. Circ. Res. 1998; 83: 141-146Crossref PubMed Scopus (171) Google Scholar), and by direct biochemical evidence for COOH terminus interactions with the cytoplasmic peptide that links domains III and IV of the α subunit (III-IV linker) (12Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (127) Google Scholar, 13Kim J. Ghosh S. Liu H. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Here we have tested the hypothesis that preservation of COOH terminus structure may also be critically important to NaV1.5 channel inactivation. Previously, we generated a structural model of the NaV1.5 C terminus based on homology to the amino-terminal lobe of calmodulin (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The model predicts six α-helices (H1-H6), the first four forming two EF-hand pairs. EF hands are helix-loop-helix motifs that typically, although not always, bind Ca2+ in the loops between helices and generally occur in pairs. One helix from each EF-hand pair is predicted to form interhelical contacts with a helix from the opposite EF hand, H1 with H4 and H2 with H3. In the present experiments, we focus on a possible role of the putative interface between H1 and H4 in stabilizing the COOH terminus structure and, in turn, in the control of channel inactivation. The predicted interface in NaV1.5 was initially of interest to us not only because several naturally occurring mutations predicted to be near it disrupt inactivation and cause multiple types of cardiac arrhythmias (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), but also because mutations in similar regions of a brain sodium channel isoform (NaV1.1) have also been reported to be linked to inherited epilepsies (15Fujiwara T. Sugawara T. Mazaki-Miyazaki E. Takahashi Y. Fukushima K. Watanabe M. Hara K. Morikawa T. Yagi K. Yamakawa K. Inoue Y. Brain. 2003; 126: 531-546Crossref PubMed Scopus (277) Google Scholar, 16Nabbout R. Gennaro E. Dalla Bernardina B. Dulac O. Madia F. Bertini E. Capovilla G. Chiron C. G. M. E. R. T. R. M. A. A. P. F. F. A. F. 2003; PubMed Scopus Google Scholar, J. G. A. I. I. P. E. A.L. J. 2004; PubMed Scopus Google Scholar). However, the importance of this interface may be more role in sodium channel because in structures for EF proteins are interactions between helices to the first and predicted helices in the NaV1.5 C terminus on the In hydrophobic are conserved this interface among EF-hand proteins R.H. Biol. PubMed Scopus Google Scholar, S. M. E. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). of the first four helices of all voltage-gated sodium channels homology in H1 and H2 and are not conserved among these channels We thus on possible interactions between helices H1 and that of hydrophobic to the interface and channel inactivation, evidence that of the COOH terminus structure the hydrophobic interface is necessary to inactivation of the NaV1.5 homology model was generated as (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). T.J. F. F. Res. PubMed Scopus Google with and for studies was on NaV1.5 in the to the by NaV1.5 and NaV1.5 with into as M. Liu H. Yang A.S. J.W. Kass R.S. J. 2004; 86: Full Text Full Text PDF PubMed Scopus Google Scholar). The used to proteins for was generated on a generated of the NaV1.5 COOH terminus in the (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The predicted proximal region was and a was the The resulting predicted to form the EF hands in the with a the coding region for and and human sodium channel α with human in cells, and with as (12Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (127) Google Scholar, M. Liu H. Yang A.S. J.W. Kass R.S. J. 2004; 86: Full Text Full Text PDF PubMed Scopus Google Scholar). In Na+ current was the The and with with The and with with The of inactivation was of to a of by a to from to In to the of Na+ was to as an Na+ was from a potential of to from to Na+ channel current was as the current or depolarization to the potential was was to Na+ channel current and as of current in with current and to the was used for and are as and proteins in to an of and was with the of and for the and in with and the and the by The NaV1.5 proteins through on was and proteins by a that the to for was to and that the the all on and by for the NaV1.5 EF was by an of on a in a a of in or the in the a of was and for between and The of the and from the with the of the as the to the and are the in the and of the and is the of was in and in are as was an was of NaV1.5 COOH a generated a structural model of the NaV1.5 COOH terminus based on homology to the amino-terminal lobe (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The model predicts a proximal region six α-helices (H1-H6), in which the first four helices are predicted to form two EF-hand pairs. One helix from each of these EF hands is predicted to form an interface with a helix from the opposite H1 with H4 and H2 with T.J. F. F. Res. PubMed Scopus Google with structural homology between the first four predicted helices of the NaV1.5 COOH terminus and helices from and studies of EF proteins from all of the helices in paired EF motifs to a hydrophobic a of hydrophobic including several that are similar among EF is predicted the interface between H1 and H4 with We thus on possible interactions between these two helices and used the of the model as as to be of critical hydrophobic as a in determining mutations that be to the interface and used inactivation to the of these on channel of predicted hydrophobic interface between the first and helices of NaV1.5 and EF-hand proteins. of predicted structural regions in this are the predicted first and the hydrophobic conserved among EF-hand proteins. structural model of NaV1.5 the first and predicted for in this are shown as with a for the importance of this interface to sodium channel function is provided by the that multiple inherited mutations of a near the interface sodium channel inactivation and cause congenital cardiac arrhythmias (8Veldkamp M.W. Viswanathan P.C. Bezzina C. Baartscheer A. Wilde A.A. Balser J.R. Circ. Res. 2000; 86: E91-E97Crossref PubMed Google Scholar, 9Bezzina C. Veldkamp M.W. van Den Berg M.P. Postma A.V. Rook M.B. Viersma J.W. van Langen I.M. Tan-Sindhunata G. Bink-Boelkens M.T. van Der Hout A.H. Mannens M.M. Wilde A.A. Circ. Res. 1999; 85: 1206-1213Crossref PubMed Scopus (557) Google Scholar, 10Rivolta I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We thus first on this of this to channel inactivation, as in an in sustained channel are in and the mutation-induced in sustained current in to depolarization is when with from wild channels of the this within the model the in of the of these for and for A.A. Mol. Biol. PubMed Scopus Google of by in a that is similar to reported changes in caused by the naturally occurring I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google and that in a in the of the for the A.A. Mol. Biol. PubMed Scopus Google We thus inactivation with possible structural of the interface and a of mutations to this hypothesis of by or in to However, with or in with a of was and in only between channels with and and with the of the The mutation, which has been linked to Brugada syndrome I. Abriel H. Tateyama M. Liu H. Memmi M. Vardas P. Napolitano C. Priori S.G. Kass R.S. J. Biol. Chem. 2001; 276: 30623-30630Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), in an in and can be to the that be to have in the that is a and hydrophobic importance to predicted to be near the of mutations sodium current in to of and inactivation from the of the to the from each and are reported as the The number in the number of in a new The of the mutations on sustained current primarily changes in inactivation that channel but we also mutation-induced changes in the of inactivation with only changes in the of The importance of the of an or in the of inactivation the that between helices H1 and in by the of an may be critical to channel and, in turn, that a putative interface may play a key role in the structural integrity of the COOH terminus of integrity of an interface is key to control of inactivation, mutations of hydrophobic predicted to form the interface be to cause similar or more in on the importance of the to the integrity of the We thus the functional consequences of of predicted to be within and critical the interhelical and and The functional consequences of the mutations are similar to that of inactivation with on channel of and in with in inactivation to changes of the not ISUS, in with the mutation, inactivation. is predicted to be more to the putative hydrophobic interface we also the by a which is a but not We found that the not However, this mutation, similar to the mutation, a in the of in of on the tested two H4 predicted to be critical to the interface within the of an and a similar on EF-hand proteins are conserved hydrophobic on the of the similar when is However, the modeling of the structure a that is more critical to the putative interhelical interface and thus predicts that of may have a on the hydrophobic interface of functional the of the We that of each of these has consequences on channel of of inactivation, as in as as the of inactivation, of of by a but not a in a but in with on inactivation and of to a not have of inactivation, but to the in a in as as changes in the of inactivation and for the is almost that for with mutations of hydrophobic in the putative the functional consequences of the are more for inactivation for with tested the of on opposite helices with by Scheuer T. Catterall W.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). changes in the of the inactivation process of mutations and a are from the of reported in The in caused by these mutations to wild is and the changes in of the inactivation process for the the in of the these are and the not the changes in of inactivation for the are or this that these changes in are on and these by Scheuer T. Catterall W.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the for the between the and the can be by the and the of inactivation is We used this and for and from the reported in and the was the We limited in that functional current but to the and both of these caused in sustained and the model to The mutations A and and A and and by both in in sustained current shown as as inactivation in the of the both mutations in in and in inactivation similar to that of the by In the the mutations in of for and for these mutations on the of inactivation, we a of inactivation for the similar to the of the changes in the However, we a of for the both mutations a The of this provide evidence that these and with in of an interhelical for an The predicted EF-hand pair in the NaV1.5 COOH terminus a which is important not only because model predicts it to be to the hydrophobic interface but also because can be used to and the of the in which it is J. Ghosh S. Liu H. Tateyama M. Kass R.S. Pitt G.S. J. Biol. Chem. 2004; 279: 45004-45012Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, C.A. PubMed Scopus (277) Google Scholar, C.A. PubMed Scopus Google Scholar). We thus a of predicted to form the EF-hand pair and as a of of this of the and provided us with of to as NaV1.5 for studies we tested the and mutations the of we to for the NaV1.5 EF and to the wild of almost all of the to be in the of the in of due to structural in to the functional of these mutations. we of We of the NaV1.5 EF and The NaV1.5 EF has an with within a hydrophobic C.A. PubMed Scopus Google Scholar). is with the of which is near the C terminus of the first helix The in a for in an of with an is to an C.A. PubMed Scopus Google Scholar). evidence that of NaV1.5 EF in the is a in which is The in are for the of the or the of the the of that the has to the that, in the of the is of the an has been in the hydrophobic of 2000; PubMed Scopus Google Scholar, J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), a has been shown to play a key role in structural In these the of the was found to be the result of interactions with In the of NaV1.5 the structural model predicts several within the with the and the to the of the in the evidence that is in a in which are to We the of in the NaV1.5 EF with in which was a Here the of a to the the of to the We used as because it is a that has limited to the for primarily through The generated from the is in The from the of this is the is the of the and is or when the is not and for C.A. PubMed Scopus (277) Google Scholar). The we found for the thus a that is with and the structural and provide biochemical evidence in of the hydrophobic interface as as the key of in and a key role of this and in the hydrophobic in channel is provided by In this a structural model as a guide, we biochemical and experiments, the of which the of a predicted EF-hand motif in the proximal COOH terminus of the NaV1.5 sodium channel and demonstrate the importance of by an interhelical hydrophobic we that of this hydrophobic interface in the of sodium channel inactivation, a novel through which sodium channel mutations may A homology model of the sodium channel COOH terminus generated by us a region in the proximal COOH terminus with homology to EF-hand (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The of EF hands in the sodium channel COOH terminus was first by J. PubMed Scopus Google Scholar). (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google and studies Balser J.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Balser J.R. Mol. Biol. 2004; 11: PubMed Scopus Google have both the and structure with the of EF-hand pairs. have not only the of the model (14Cormier J.W. Rivolta I. Tateyama M. Yang A.S. Kass R.S. J. Biol. Chem. 2002; 277: 9233-9241Abstract Full Text Full Text PDF PubMed Scopus (119) Google but also of between on two of the helices and to the present M. Liu H. Yang A.S. J.W. Kass R.S. J. 2004; 86: Full Text Full Text PDF PubMed Scopus Google Scholar). In generated on the similar to particularly in the predicted helices H1 and the model was generated for the COOH terminus of the cardiac sodium channel this region is conserved among several voltage-gated sodium channels, particularly within the predicted first and helices and several disease-associated mutations that cause inherited epilepsies have been reported in this region of the brain sodium channels (15Fujiwara T. Sugawara T. Mazaki-Miyazaki E. Takahashi Y. Fukushima K. Watanabe M. Hara K. Morikawa T. Yagi K. Yamakawa K. Inoue Y. Brain. 2003; 126: 531-546Crossref PubMed Scopus (277) Google Scholar, 16Nabbout R. Gennaro E. Dalla Bernardina B. Dulac O. Madia F. Bertini E. Capovilla G. Chiron C. G. M. E. R. T. R. M. A. A. P. F. F. A. F. 2003; PubMed Scopus Google Scholar, J. G. A. I. I. P. E. A.L. J. 2004; PubMed Scopus Google Scholar), and, the mutations in NaV1.5, these mutations also disrupt inactivation, the that a putative functional role of this region with to channel may be conserved among voltage-gated sodium The EF-hand pair is a occurring structure helices to form a hydrophobic by on the of the in but also may in interactions that function homology model predicts a structure with EF-hand hydrophobic conserved among EF-hand proteins and conserved in model of the NaV1.5 C terminus have been shown to play functional in proteins. studies have the hydrophobic of EF-hand proteins and have functional including on Ca2+ as as in T. K. A. T. S. 2001; PubMed Scopus Google Scholar, C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, K. E. S. 1998; PubMed Scopus Google Scholar, Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that of the interface inactivation and sustained Na+ channel current most with mutations that the of the of the proximal EF-hand in the COOH terminus of the sodium channel have a on inactivation of inactivation is on interactions between the NaV1.5 cytoplasmic linker and the COOH terminus and this is through the predicted α helix of the COOH terminus (12Motoike H.K. Liu H. Glaaser I.W. Yang A.S. Tateyama M. Kass R.S. J. Gen. Physiol. 2004; 123: 155-165Crossref PubMed Scopus (127) Google Scholar). studies demonstrate that with the EF-hand Balser J.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). may be possible that the linker region in the COOH terminus is to the EF-hand terminus and inactivation gating. is that of the EF in the proximal COOH terminus a of predicted by the model of the COOH terminus that is to of the sodium altered cause the in that we A is that of by the hydrophobic interface is necessary to interactions between the sodium channel and proteins. The COOH terminus of the sodium channel has been shown to with a number of proteins that can or H. Kass R.S. 2005; PubMed Scopus Google Scholar). In a region H4 has been shown to with the region of the and a in that this has been shown to cause J. G. A. I. I. P. E. A.L. J. 2004; PubMed Scopus Google Scholar). paired EF-hand motifs have been in several and interactions S. 1999; PubMed Scopus Google Scholar, B. C. J. and T. Scholar, 2001; PubMed Scopus Google Scholar, S. A. K. Y. 2003; PubMed Scopus Google Scholar, Pitt G.S. K. H. 1999; PubMed Scopus Google Scholar), including of voltage-gated sodium this the that of the changes by of the hydrophobic interface be the result of on in to or more direct on caused by of the hydrophobic of the EF-hand the of inactivation to mutations that disrupt this hydrophobic of COOH terminus structure in the of inactivation that from between and/or In this that hydrophobic within a predicted EF-hand motif play a critical role in sodium channel inactivation and a structural basis for the altered function of disease-linked mutations in the NaV1.5 COOH terminus as as sodium channel We for for and S. Pitt for the of with